Indicator for the determination of reduced pyridine coenzymes

ABSTRACT

REDUCED PYRIDINE COENZYMES, SUCH AS NICOTIMAMIDEADENINE-DINUCLEOTIDDE (NADH) AND NICOTINAMIDE-ADENINEDINUCLEOTIDE PHOSPHATE (NADPH), ARE CONVENIENTLY DETERMINED COLORIMETRICALLY WITH A STABLE AND INHERENTLY COLOR-PRODUCING INDICATOR COMPRISING A BENZOPHENOXAZINE OR BENZOPHENOTHIAZINE COMPOUND OF THE FORMULA: 1-(R=),3,4-(-X-(2,1-NAPHTHYLENE(-Y))-N=)-CYCLOHEXA-2,5-DIENE WHEREIN R IS OXYGEN OR THE GROUP R1-N(-R2)= IN WHICH R1 AND R2 ARE HYDROGEN OR LOWER ALKYL OF FROM 1 TO 6 CARBON ATOMS; X IS SULFUR OR OXYGEN; AND Y IS HYDROGEN OR HYDROXYL.

United States Patent 3,713,986 INDICATOR FOR THE DETERMINATION OF REDUCED PYRlDlNE COENZYMES Hans Ulrich Bergmeyer, Erich Haid, and Mchael Nelboeck-Hochstetter, Tutzing, Upper Bavaria, and Gunter Weimann, Percha, Germany, assignors to Boehringer Mannheim GmbH, Postfach, Germany No Drawing. Filed Nov. 17, 1970, Ser. No. 90,467 Claims priority, application Germany, Nov. 26, 1969, P 19 59 410.6 Int. Cl. (312k 1/04 U.S. Cl. 195-103.5 R 18 Claims ABSTRACT OF THE DISCLOSURE Reduced pyridine coenzymes, such as nicotinamideadenine-dinucleotide (NADH) and nicotinamide-adeninedinucleotide phosphate (NADPH), are conveniently determined colorimetrically with a stable and inherently color-producing indicator comprising a benzophenoxazine or benzophenothiazine compound of the formula:

in which R and R are hydrogen or lower alkyl of from 1 to 6 carbon atoms; X is sulfur or oxygen; and Y is hydrogen or hydroxyl.

The present invention is concerned with a colorimetric method for the determination of reduced pyridine coenzymes, such as nicotinamide adenine dinucleotide (NADH) or nicotinamide-adenine-dinucleotide phosphate (NADPH).

The reduced pyridine coenzymes are well-known indicators in analytical biochemistry. With their use, it is possible to determine a wide variety of metabolic products in the animal and vegetable kingdoms. Furthermore, in recent times, the determinations of enzymes which are possible with the use of these pyridine coenzymes have achieved an enormous importance in the diagnosis of damage to or disfunction of organs in human and veterinary medicine.

The measurement principle in question depends upon the varying light absorption of the oxidized and reduced pyridine coenzyme (NAD, NADH, NADP, NADPH) in ultra-violet light. However, such measurements necessitate the use of complicated and expensive devices so that the applicability of these methods of measurement is very limited. In order to overcome this disadvantage, attempts have been made to make directly visible the redox reactions which occur, with the help of dyestuffs. Hitherto, however, it has only been possible to couple the reduced inc pyridine coenzyme with dyestuffs via the catalyst phenazine-methosulfate (which is also known as N-methylphenazonium methosulfate) or via the enzyme diaphorase. However, these two indicators also suffer from considerable disadvantages, the overcoming of which is of greater interest.

Thus, the stability of these known indicators is insuflicient. Phenazine-methosulfate (PMS), for example, loses -80% of its effectiveness within the course of 14 days by the action of light. Diaphorase, as an enzyme, also has only a very limited storage stability, especially at ambient temperature. Furthermore, diaphorase is very expensive. In addition, both indicators suffer from the disadvantage that they themselves do not exhibit a color change which can be used for measurement but are only effective as electron carriers to a tetrazolium salt which is thereby reduced with the formation of a colored formazane. The reaction which takes place can, in the case of an enzymatic test, be represented by the following equation:

-I- enzyme Substrate (reduced) NAD Substrate (oxidized) NADH 11+ NADH PMS (oxidized) NAD+ l- PMS (reduced) PMS (reduced) TTC --a PMS (oxidized) formazane 1 TTC-tetrazolium salt.

in which R and R which may be the same or different, are hydrogen atoms or lower alkyl radicals containing up to 6 carbon atoms; X is sulfur or oxygen and Y is hydrogen or hydroxyl; and used as indicators for the determination of the reduced pyridine coenzymes.

The preferred compounds of the above-given general formula are those in which R and R are methyl and X is oxygen.

The compounds to be used according to the present invention are themselves colored and thus permit the direct determination of the reduced pyridine coenzymes since they are decolorised by them. Thus, it is possible simply to titrate the reduced pyridine coenzymes with solutions of the indicators to be used according to the present invention. In this way, a determination of the reduced pyridine coenzymes is possible with only a small expenditure for apparatus.

However, the compounds to be used according to the present invention can also act as electron carriers to tetrazolium salts with the formation of coloured formazanes and can, therefore be used in the same manner as the two above-mentioned known electron carriers. However, in camparison with these two known compounds, they have the advantage of a better stability and, in particular, they can also be stored at ambient temperature for long periods of time.

The compounds to be used according to the present invention are characterized by the presence of a benzene ring in the 2,3- or 2,1-position of the phenoxazine or phenothiazine ring system. This fused benzene ring can be substituted by a hydroxyl group, which is preferably in the 2'- or 3'- position. Preferred compounds to be used according to the present invention are 7-dimethylamino-1,Z-benzophenoxazine (Meldolblue), 7-dimethylamino-2-hydroxy-1,2-benzophenoxazine, 7-dimethylamino-3-hydroxy-1,2-benzophenoxazine, 7-amino-1,2 benzophenoxazine,

2,3-benzophenoxazine and 7-dimethylamino-1,2-benzophenothiazine.

The compounds to be used according to the present invention are known. The amounts used depend upon whether there is to be carried out a direct determination by decolorisation or whether the compound is to be used as a carrier to a tetrazolium salt. In the first case, it is a matter of a stoichiometric reaction and there must, therefore, be used stoichiometric amounts, referred to the reduced pyridine coenzyme. In the latter case, it is a question of a catalytic action, in which the compounds act as electron carriers. In this case, it is suflicient to use very small amounts of the compounds.

Some of the compounds to be used according to the present invention are water-soluble, whereas others are Water-insoluble. The water-insoluble compounds are used dissolved in aqueous or pure alcohols, preferably in isopropanol.

When carrying out the indicator reaction with the addition of a tetrazolium salt, there can be used the tetrazolium salts which have been previously employed for this purpose. Those which have proved to be particularly useful are 3-(4,5-dimethyl-thiazo1yl-2)-2,5-diphenyl-tetrazolium bromide and iodonitrotetrazolium chloride.

In the case of carrying out a direct determination in the absence of a tetrazolium salt, it is expedient to work with the exclusion of atmospheric oxygen in order to avoid a reoxidation of the leuco dyestuffs by atmospheric oxygen. Thus, for example, in the case of a titration, as reaction vessel there is used a closed vessel which is flushed with an inert gas.

The compounds to be used according to the present invention can, as a result of their stability, also be employed, for example, when applied to carrier materials, such as paper strips, gel layers and the like, as indicators for the determination of pyridine coenzyme activities.

The following examples are given for the purpose of illustrating the present invention:

EXAMPLE 1 Titrimetric determination of NADH Reagents:

0.01% solution of 7-dimethylamino-1,2-benzophenoxazine (Meldolblue) nitrogen bomb NADH standard solution Method: 10 ml. distilled water were slowly stirred with a magnetic stirrer in a 50 m1. 3-necked round-bottomed flask. The atmospheric oxygen present in the flask was flushed out by a continuously flowing current of nitrogen from the nitrogen bomb. Through a tube provided for the flow off of the nitrogen, the NADH solution to be measured was added by means of a thin pipette to the interior of the flask. Using a burette, which was connected to the flask by a ground-glass joint, the blue-colored solution of Meldolblue was added dropwise into the flask, decoloration of this solution thereby taking place immediately. As soon as the Meldolblue solution dropped in was no longer decolorized, the end point of the titration was reached. The amount of Meldolblue solution used was a measure for the NADH content of the solution pipetted in.

Without removal of the contents of the flask, several measurements could be carried out one after the other.

In order to obtain a precise measurement, it is recommended to calibrate the Meldolblue solution with a standardized NADH solution.

Calculation:

Mg. NADH in the standard soln. used Xml. Meldolblue soln. used ml. Meldolblue soln. used for NADH standard :mg. NADH in the unknown soln.

EXAMPLE 2 The compounds set out in the following table were used for the determination of the activity of lactate dehydrogenase, phenazine-methosulfate (PMS) being used as comparison compound.

The determinations were carried out in the following manner:

1.78 ml. glycyl-glycine buffer; pH 8.5; 0.1 molar 0.20 ml. 1-lactate;c.=10 mg./ml.

0.20 ml. NAD; c.=10 mg./ml.

0.50 ml. 3-(4,5-dimethiazolyl-2)-2,S-diphenyl-tetrazolium bromide solution; c.=0.5 mg./ ml.

0.30 ml. electron carrier according to table; c.=0.1 mg./

The test was commenced with 0.02 ml. purified lactate dehydrogenase solution (0.068 IU).

After 10 minutes, the extinction was measured at 578 m in a photometer (Eppendorf). The following table gives the results obtained:

7 dimethylamino 2' hydroxy 1,2 benzophenoxazonium chloride 0.501 7 dimethylamino 3' hydroxy 1,2 benzophenoxazonium chloride 0.278 7-dimethylamino-phenothiazoniumchloride 0.285 1,2 benzophenoxaz 7 one 0.200 3 hydroxy 1,2 benzophenazin-7-one 0.151 Phenazine methosulphate (comparison) 0.524

The results show that the compounds to be used according to the present invention were suitable as indicators for reduced pyridine coenzymes in the same way as the know PMS.

Since, in the case of enzyme measurements, the activity depends upon the concentration of the indicator used, it each test series, an enzyme standard was also measured and the value obtained referred thereto.

It will be understood that the foregoing specifications and examples are illustrative and not limitative of the present invention in that many other embodiments of the invention will suggest themselves to those skilled in the art.

What is claimed is:

1. Method for the determination of reduced pyridine coenzymes which comprises contacting a test mixture with an indicator comprising a compound of the general formula:

in which R and R are hydrogen or lower alkyl of tfrom 1 to 6 carbon atoms; X is sulfur or oxygen; and Y is hydrogen or hydroxyl.

2. Method as claimed in claim 1 wherein R is oxygen, 3. Method as claimed in claim 1 wherein R is 9. Method as claimed in claim 1 wherein said compound is 7-amino-1,2-benzophenoxazonium chloride.

10. Method as claimed in claim 1 wherein said compound is 7-dimethylamino-2'-hydroxy-1,2-benzophenoxazoniunm chloride.

11. Method as claimed in claim 1 wherein said compound is 7 dimethylamino 3' hydroxy 1,2-benzophenoxazonium chloride.

12. Method as claimed in claim 1 wherein said compound is 7-dimethylamino-phenothiazonium chloride.

13. Method as claimed in claim 1 wherein a solution containing a reduced pyridine coenzyme is titrated with a solution of said compound.

14. Method as claimed in claim 13 wherein the titration is carried out in the absence of atmospheric oxygen.

15. Method as claimed in claim 1 wherein said indicator comprises, additionally, a tetrazolium salt.

16. Method as claimed in claim 15 wherein the tetrazolium salt is 3 (4,5 dimethyl thiazolyl 2)-2,5- diphenyl-tetrazolium bromide or iodonitrotetrazolium chloride.

17. Composition for the colorimetric determination of reduced pyridine coenzymes which composition comprises as an indicator a compound as recited in claim 1 and, in addition to said compound, a tetrazolium salt.

18. Composition as claimed in claim 17 wherein the tetrazolium salt is 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl-tetrazolium bromide or iodonitrotetrazolium chloride.

References Cited George Guilbault, Enzymatic Methods of Analysis, 1st ed.; 1970, Pergamon Press; pp. 92 and 93.

A. LOUIS MONACELL, Primary Examiner R. J. WARDEN, Assistant Examiner US. Cl. X.R. 23230 B 

